1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
2.1. Root Development
2.2. Genes Regulating Root Structure
2.3. Geortropism
3. SUMMARY OF THE INVENTION
3.1. Definitions
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. SCR Genes
5.1.1. Isolation of SCR Genes
5.1.2. Expression of SCR Gene Products
5.1.3. Antibodies to SCR Proteins and Polypeptides
5.1.4. SCR Gene of Gene Products as Markers for Qualitative Trait Loci
5.2. SCR Promoters
5.2.1. Cis-Regulatory Elements or SCR Promoters
5.2.2. SCR Promoter-Driven Expression Vectors
5.3. Production of Transgenic Plants and Plant Cells
5.3.1. Transgenic Plants that Ectopically Express SCR
5.3.2. Transgenic Plants that Suppress Endogenous SCR Expression
5.3.3. Transgenic Plants that Express a Transgene Controlled by the SCR Promoter
5.3.4. Screening of Transformed Plants for Those Having Desired Altered Traits
EXAMPLE 1: Arabidopsis SCR Gene
6.1 Material and Methods
6.1.1. Plant Culture
6.1.2. Genetic Analysis
6.1.3. Mapping
6.1.4. Phenotypic Analysis
6.1.5. Molecular Techniques
6.1.6. In Situ Hybridization
6.2. Results
6.2.1. Characterization of the SCR Phenotype.
6.2.2. Characterization of Cell Identify in SCR Roots
6.2.3. Molecular Cloning of the SCR Gene
6.2.4. The SCR Gene has Motifs that Indicate it is a Transcription Factor
6.2.5. SCR is a Member of a Novel Protein Family
6.2.6. SCR is Expressed in the Cortex/Endodermal Initials and in the Endodermis
6.3. Discussion
6.3.1. The SCR Gene Regulates an Asymmetric Division Required for Root Radial Organization
6.3.2. SCR Involvement in Cell Specification of Cell Division
6.3.3. A Role for SCR in Embryonic Development
6.3.4. Tissue-Specific Expression of SCR is Regulated at the Transcriptional Level
6.3.5. A new Family of Transcriptional Regulators
7. EXAMPLE 2: Enhancer Trap Analysis of Root Development
7.1. Material and Methods
7.1.1. Plant Growth Conditions
7.1.2. Histology and Gus Staining
7.1.3. Construction of Enhancer Trap Lines
7.2. Results
7.2.1. Differential in the LRP
7.2.2. Marker Lines
7.2.3. ET199 Provides Evidence for the Role of SCR in Plant Development
8. EXAMPLE 3: Activity of Arabidopsis SCR Promoter in Transgenic Roots
9. EXAMPLE 4: Isolation SCR Sequences Using PRC-Cloning Strategy
10. EXAMPLE 5: Expression Pattern of Maize ZCR Gene in Root Tissue
11. EXAMPLE 6: Expression Pattern of ZCR Gene in Soybean Roots and Root Nodules
12. EXAPMPLE 7: SCR Expression Affects Gravitropism of Aerial Structures
13. Deposit of Microorganisms
The present invention generally relates to the SCARECROW (SCR) gene family and their promoters. The invention more particularly relates to ectopic expression of members of the SCARECROW gene family in transgenic plants to artificially modify plant structures. The invention also relates to utilization of SCARECROW promoter for tissue and organ specific expression of heterologous gene products.
Asymmetric cell divisions, in which a cell divides to give two daughters with different fates, play an important role in the development of all multicellular organisms. In plants, because there is no cell migration, the regulation of asymmetric cell divisions is of heightened importance in determining organ morphology. In contrast to animal embryogenesis, most plant organs are not formed during embryogenesis. Rather, cells that form the apical meristems are set aside at the shoot and root poles. These reservoirs of stem cells are considered to be the source of all post-embryonic organ development in plants. A fundamental question in developmental biology is how meristems function to generate plant organs.
2.1. Root Development
Root organization is established during embryogenesis. This organization is propagated during postembryonic development by the root meristem. Following germination, the development of the postembryonic root is a continuous process, a series of initials or stem cells continuously divide to perpetuate the pattern established in the embryonic root (Steeves and Sussex, 1972, Patterns in Plant Development, Englewood Cliffs, N.J.: Prentice-Hall, Inc.).
Due to the organization of the Arabidopsis root it is possible to follow the fate of cells from the meristem to maturity and identify the progenitors of each cell type (Dolan et al., 1993, Development 119:71-84). The Arabidopsis root is a relatively simple and well characterized organ. The radial organization of the mature tissues in the Arabidopsis root has been likened to tree rings with the epidermis, cortex, endodermis and pericycle forming radially symmetric cell layers that surround the vascular cylinder (FIG. 1A). See also Dolan et al., 1993, Development 20 119:71-84. These mature tissues are derived from four sets of stem cells or initials: i) the columella root cap initial; ii) the pericycle/vascular initial; iii) the epidermal/lateral root cap initial; and iv) the cortex/endodermal initial (Dolan et al., 1993, Development 119:71-84). It has been shown that these initials undergo asymmetric divisions (Scheres et al., 1995, Development 121:53-62). The cortex/endodermal initial, for example, first divides anticlinally (in a transverse orientation) (FIG. 1B). This asymmetric division produces another initial and a daughter cell. The daughter cell, in turn, expands and then divides periclinally (in the longitudinal orientation) (FIG. 1B). This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG. 1B). 2.2. Genes Regulating Root Structure
Mutations that disrupt the asymmetric divisions of the cortex/endodermal initial have been identified and characterized (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). short-root (shr) and scarecrow (scr) mutants are missing a cell layer between the epidermis and the pericycle. In both types of mutants the cortex/endodermal initial divides anticlinally, but the subsequent periclinal division that increases the number of cell layers does not take place (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). The defect is first apparent in the embryo and it extends throughout the entire embryonic axis which includes the embryonic root and hypocotyl (Scheres et al., 1995, Development 121:53-62). This is also true for the other radial organization mutants characterized to date, suggesting that radial patterning that occurs during embryonic development may influence the post-embryonic pattern generated by the meristematic initials (Scheres et al., 1995, Development 121:53-62).
Characterization of the mutant cell layer in shr indicated that two endodermal-specific markers were absent (Benfey et al., 1993, Development 119:57-70). This provided evidence that the wild-type SHR gene may be involved in specification of endodermis identity.
2.3. Geotropism
In plants, the capacity for gravitropism has been correlated with the presence of amyloplast sedimentation. See, e.g., Volkmann and Sievers, 1979, Encyclopedia Plant Physiol., N.S. vol 7, pp. 573-600; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Bjxc3x6rkmann, 1992, Adv. Space Res. 12:195-201; Poff et al., in The Physiology of Tropisms, Meyerowitz and Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962. Amyloplast sedimentation only occurs in cells in specific locations at distinct developmental stages. That is, when and where sedimentation occurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol. 127:193-252). In roots, amyloplast sedimentation only occurs in the central (columella) cells of the rootcap; as these cells mature into peripheral cap cells, the amyloplasts no longer sediment (Sack and Kiss, 1989, Amer. J. Bot. 76:454-464; Sievers and Braun, in The Root Cap: Structure and Function, Wassail et al. (eds.), New York: M. Dekker (1996) pp. 31-49). In stems of many plants, including Arabidopsis, amyloplast sedimentation occurs in the starch sheath (endodermis) especially in elongating regions of the stem (von Guttenberg, Die Physiologischen Scheiden, Handbuch der Pflanzenanatomie;
K. Linsbauer (ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217; Sack, 1987, Can. J. Bot. 65:1514-1519; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Caspar and Pickard, 1989, Planta 177:185-197; Volkmann et al., 1993,J. Pl. Physiol. 142:710-6).
Gravitropic mutants have been studied for evidence that proves the role of amyloplast sedimentation in gravity sensing. However, many gravitropic mutations affect downstream events such as auxin sensitivity or metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seem to affect gene products that process information from gravity sensing. For example, the lazy mutants of higher plants and comparable mutants in mosses can clearly sense and respond to gravity, but the mutations reverse the normal polarity of the gravitropic response (Gaiser and Lomax, 1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant Cell Environ 9:637-644). Other mutations appear to affect gravitropism of specific organs. For example, sgr mutants have defective shoot gravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki et al., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res. 109:129-137).
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in certain tissues of roots and stems. SCR expression controls cell division of certain cell types in roots, and affects the organization of root and stem. The invention relates to the SCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and its orthologs and paralogs), SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR regulatory regions and the use of the foregoing to improve agronomically valuable plants.
The invention is based, in part, on the discovery, identification and cloning of the gene responsible for the scarecrow phenotype. In contrast to the prevailing view that the SCR gene was likely to be involved in the specification of endodermis, the inventors have determined that the mutant cell layer in roots of scr mutants has differentiated characteristics of both cortex and endodermis. This is consistent with a role for SCR in the regulation of the asymmetric cell division rather than in specification of the identity of either cortex or endodermis. The inventors have also determined that SCR expression affects the gravitropism of plant aerial structures such as the stem.
One aspect of the invention relates to the heterologous expression of SCR genes and related nucleotide sequences, and specifically the Arabidopsis SCR genes, in stably transformed higher plant species. Modulation of SCR expression levels can be used to advantageously modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants.
Another aspect of the invention relates to the use of promoters of SCR genes, and specifically the use of Arabidopsis SCR promoter to control the expression of protein and RNA products in plants. Plant SCR promoters have a variety of uses, including but not limited to expressing heterologous genes in the embryo, root, root nodule, and stem of transformed plants.
The invention is illustrated by working examples described infra which demonstrate the isolation of the Arabidopsis SCR gene using insertion mutagenesis. More specifically, T-DNA tagging of genomic and cDNA clones of the Arabidopsis SCR gene are described. Additional working examples include the isolation of SCR sequences from plant genomes using PCR amplification in combination with screening of genomic libraries, and heterologous gene expression in transgenic plants using SCR promoter expression constructs.
Structural analysis of the deduced amino acid sequence of Arabidopsis SCR protein indicates that SCR encodes a transcription factor. Northern analysis, in situ hybridization analysis and enhancer trap analysis show highly localized expression of Arabidopsis SCR in embryos and roots. Genetic analysis shows SCR expression also affects gravitropism of aerial structures (e.g., stems). This indicates that SCR is also expressed in those structures.
Computer analysis of the deduced amino acid sequence of Arabidopsis SCR protein with those of Expressed Sequence Tag (EST) sequences in GenBank reveals the existence of at least thirteen SCR genes in Arabidopsis, one SCR gene in maize, four SCR genes in rice, and one SCR gene in Brassica. A further aspect of the invention relates to the use of such EST sequences to obtain larger and/or complete clones of the corresponding SCR gene.
The various embodiments of the claimed invention presented herein are by the way of illustration and are not meant to limit the invention.
3.1. Definitions
As used herein, the terms listed below will have the meanings indicated.
35S=cauliflower mosaic virus promoter for the 35S transcript
cDNA=complementary DNA
cis-regulatory element=A promoter sequence 5xe2x80x2 upstream of the TATA box that confers specific regulatory response to a promoter containing such an element. A promoter may contain one or more cis-regulatory elements, each responsible for a particular regulatory response
coding sequence=sequence that encodes a complete or partial gene product (e.g., a complete protein or a fragment thereof)
DNA=deoxyribonucleic acid
EST=expression tagged
functional portion=a functional portion of a promoter is any portion of a promoter that is capable of causing transcription of a linked gene sequence, e.g., a truncated promoter
gene fusion=a gene construct comprising a promoter operably linked to a heterologous gene, wherein said promoter controls the transcription of the heterologous gene
gene product=the RNA or protein encoded by a gene sequence
gene sequence=sequence that encodes a complete gene product (e.g., a complete protein)
GUS=1,3-xcex2-Glucuronidase
gDNA=genomic DNA
heterologous gene=In the context of gene constructs, a heterologous gene means that the gene is linked to a promoter that said gene is not naturally linked to. The heterologous gene may or may not be from the organism contributing said promoter. The heterologous gene may encode messenger RNA (mRNA), antisense RNA or ribozymes
homologous promoter=a native promoter of a gene that selectively hybridizes to the sequence of a SCR gene described herein
mRNA=messenger RNA
operably linked=A linkage between a promoter and gene sequence such that the transcription of said gene sequence is controlled by said promoter
ortholog=related gene in a different plant (e.g., maize ZCARECROW gene is an ortholog of the Arabidopsis SCR gene)
paralog=related gene in the same plant (e.g., Arabidopsis SRPa1 is a paralog of Arabidopsis SCR gene)
RNA=ribonucleic acid
RNase=ribonuclease
SCR=SCARECROW gene or gene product, encompasses (italic) SCR and ZCR genes and their orthologs and paralogs
SCR=SCARECROW protein
scr=scarecrow mutant (e.g., scr1) (lower case)
ZCR=maize ZCARECROW gene, a paralog of, for example, the Arabidopsis SCR gene
SCR protein means a protein containing sequences or a domain substantially similar to one or more motifs (i.e., Motif I-VI), preferably MOTIF III (amino acid residues 373-435 of SEQ ID NO:2) (VHIID), (amino acid residues 8-12 of SEQ ID NO:12) of Arabidopsis SCR protein as shown in FIGS. 13A-F and FIGS. 15A-S. SCR proteins include SCR ortholog and paralog proteins having the structure and activities described herein.
SCR polypeptides and peptides include deleted or truncated forms of the SCR protein, and fragments corresponding to the SCR motifs described herein.
SCR fusion proteins encompass proteins in which the SCR protein or an SCR polypeptide or peptide is fused to a heterologous protein, polypeptide or peptide.
SCR gene, nucleotides or coding sequences means nucleotides, e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides or peptides, or SCR fusion proteins.
SCR gene products include transcriptional products such as mRNAs, antisense and ribozyme molecules, as well as translational products of the SCR nucleotides described herein including but not limited to the SCR protein, polypeptides, peptides and/or SCR fusion proteins.
SCR promoter means the regulatory region native to the SCR gene in a variety of species, which promotes the organ and tissue specific pattern of SCR expression described herein.
FIGS. 1A-B. Schematic of Arabidopsis root anatomy. FIG. 1A. Transverse section showing the four tissues, epidermis, cortex, endodermis and pericycle that surround the vascular tissue. In the longitudinal section, the epidermal/lateral root cap initials and the cortex/endodermal initials are shown at the base of their respective cell files. FIG. 1B. Schematic of division pattern of the cortex/endodermal initial. The initial expands then divides anticlinally to reproduce itself and a daughter cell. The daughter then divides periclinally to produce the progenitors of the endodermis and cortex cell lineages. Abbreviations: C, cortex; Da, daughter cell; E, endodermis; In, initial.
FIGS. 2A-F. Phenotype of scr mutant plants. FIG. 2A. Shown left to right are 12-day scr-2, scr-1 and wild-type seedlings grown vertically on nutrient agar medium. FIG. 2B. 21-day scr-2 mutant plants in soil. FIG. 2C. Transverse section through primary root of 7-day scr-2. FIG. 2D. Transverse section through primary root of 7-day wild-type (WT). FIG. 2E. Transverse section through lateral root of 12-day scr-1 mutant seedling. FIG. 2F. Transverse section through root regenerated from scr-1 callus. Bar, 50 xcexcm. Abbreviations: C, cortex; En, endodermis; Ep, epidermis; M, mutant cell layer; P, pericycle; V, vascular tissue.
FIGS. 3A-F. Characterization of the cellular identity of the mutant cell layer. FIG. 3A. Endodermis-specific Casparian band staining of transverse sections through the primary root of 7-day scr-1 mutant. (Note: the histochemical stain also reveals xylem cells in the vascular cylinder.) FIG. 3B. Casparian band staining of transverse sections through the primary root of 7-day wild-type (WT). FIG. 3C. Immunostaining with the endodermis (and a subset of vascular tissue) specific JIM13 monoclonal antibodies on transverse root sections of scr-2 mutant. FIG. 3D. Immunostaining with JIM13 monoclonal antibodies on transverse root sections of WT. FIG. 3E. Immunostaining with the JIM7 monoclonal antibody that stains all cell walls on transverse root sections of scr-2 mutant. FIG. 3F. Immunostaining with JIM7 monoclonal antibodies on transverse root sections of WT. Bar, 25 xcexcm. Abbreviations are same as those for description of FIGS. 2A-2F and: Ca, casparian strip.
FIGS. 4A-F. Immunostaining. FIG. 4A. Immunostaining with the cortex (and epidermis) specific CCRC-M2 monoclonal antibodies on transverse root sections of scr-1 mutant. FIG. 4B. Immunostaining with CCRC-M2 antibodies on transverse root sections of scr-2 mutant. FIG. 3C. Immunostaining with CCRC-M2 antibodies on transverse root sections of wild-type (WT). FIG. 4D. Immunostaining with the CCRC-M1 monoclonal antibodies (specific to a cell wall epitope found on all cells) on transverse root sections of scr-1. FIG. 4E. Immunostaining with CCRC-M1 antibodies on transverse root sections of scr-2. FIG. 4F. Immunostaining with CCRC-M1 antibodies on transverse root sections of WT. Bar, 30 xcexcm. Abbreviations are same as those for description of FIGS. 2A-2F.
FIGS. 5A-E. Structure of the Arabidopsis SCARECROW gene. FIG. 5A. Nucleic acid sequence and deduced amino acid sequence of the Arabidopsis SCR genomic region (SEQ ID NO:1) and (SEQ ID NO:2), respectively. Regulatory sequences including: (i) TATA box, (ii) ATG start codon, and (iii) potential polyadenylation sequence are underlined. Within the deduced amino acid sequence homopolymeric repeats are underlined. FIG. 5B. Schematic diagram of genomic clone indicating possible functional motifs, T-DNA insertion sites and subclones used as probes. Abbreviations: Q,S,P,T, region with homopolymeric repeats of these amino acids; b, region with similarity to the basic region of bZIP factors; I and II, regions with leucine heptad repeats; E, acidic region. FIG. 5C. Comparison of the charged region found in Arabidopsis SCR protein with that found in bZIP transcription factors, SCR bZIP-like domain (SEQ ID NO:3), GCN4 (SEQ ID NO:4), TGA1 (SEQ ID NO:5), C-Fos (SEQ ID NO:6), c-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2 (SEQ ID NO:10), RAF-1 (SEQ ID NO:11). FIG. 5D. Translations of EST clones encoding putative peptide having similarities to the VHIID domain region of Arabidopsis SCR protein (SEQ ID NO:12), F13896 (SEQ ID NO:13), Z37192 (SEQ ID NO:14), and Z25645 (SEQ ID NO:15) are from Arabidopsis, T18310 (SEQ ID NO:17) is from maize and D41474 (SEQ ID NO:16) is from rice. FIG. 5E. The deduced amino acid sequence of the Arabidopsis SCARECROW gene (SEQ ID NO:2).
FIGS. 6A-B. Expression of the Arabidopsis SCARECROW gene. FIG. 6A. Northern blot of total RNA from wild-type siliques (Si), roots (R), leaves (L) and whole seedlings (Sd) hybridized with Arabidopsis SCR probe a and with a probe from the Arabidopsis glutamine dehydrogenase (GDH) gene (Melo-oliveira et al., 1996, Proc. Natl. Acad. Sci. USA 93:4718-4723) as a control for RNA integrity. (GDH expression is lower in siliques than in vegetative tissues.) The 1.6 kb band corresponds to the GDH gene and the approximately 2.5 kb band corresponds to SCR. Ribosomal RNA is shown as a loading control. FIG. 6B. Northern blot of Arabidopsis wild-type, scr-1 and scr-2 total RNA, probed with Arabidopsis SCR probe xe2x80x9caxe2x80x9d corresponding to a cDNA sequence shown in FIG. 5B, and with the GDH probe. In scr-2 mutant additional bands of 4.1 kb and 5.0 kb were detected.
FIGS. 7A-G. In situ hybridization and enhancer trap analyses of Arabidopsis SCR expression. FIG. 7A. SCR RNA expression detected by in situ hybridization of SCR antisense probe to a longitudinal section through the root meristem. FIG. 7B. In situ hybridization of SCR antisense probe to a transverse section in the meristematic region. FIG. 7C. In situ hybridization of SCR antisense probe to late torpedo stage embryo. FIG. 7D. Negative control in situ hybridization using a SCR sense probe to a longitudinal section through the root meristem. FIG. 7E. GUS expression in a whole mount in the enhancer trap line, ET199 in primary root tip. FIG. 7F. GUS expression in the ET199 line in transverse root section in the meristematic region. FIG. 7G. GUS expression in ET199 detected in a section through the root meristem. GUS expression is observed in the cortex/endodermal initial, and in the first cell in the endodermal cell lineage but not in the first cell of the cortex lineage. Expression in two endodermal layers is observed higher up in the root because the section was not median at that point. Bar, 50 xcexcm. Abbreviations are same as those in the description of FIGS. 2A-2F.
FIG. 8. Partial nucleotide sequence (SEQ ID NO:18) and deduced amino acid sequence (SEQ ID NO:19) of the Arabidopsis SRPa4 gene.
FIG. 9. Partial nucleotide sequence (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) of the Arabidopsis SRPa3 gene.
FIG. 10. Partial nucleotide sequence (SEQ ID NO22) of the Arabidopsis SRPa1 gene.
FIG. 11A. Nucleotide sequence (SEQ ID NO:24) and deduced amino acid sequence (SEQ ID NO:25) of the maize Zm-Scl1 fragment.
FIG. 11B. Partial nucleotide sequence (SEQ ID NO:25) and deduced amino acid sequence (SEQ ID NO:26) of the maize SRPm1 gene (Zm-Scl2).
FIGS. 12A-B. Nucleotide sequence of rice SRPo3 EST clone. FIG. 12A. Sequence of 5xe2x80x2 end of EST clone (SEQ ID NO:28). FIG. 12B. Sequence of 3xe2x80x2 end of EST clone (SEQ ID NO:29).
FIGS. 13A-F. Comparison of the amino acid sequence of members of the SCARECROW family of genes. Conserved Motifs I through VI are indicated by dashed line above the aligned sequences. Consensus sequences are shown in bold. See Table 1 for the identity and sequence identifier number of each of the sequences shown in this Figure. Hu-scr-1=Human SCR paralog (SEQ ID NO:40).
FIG. 14. Restriction map of the approximately 8.8 kb Eco RI insert DNA of lambda clone, t643, containing the Arabidopsis SCR gene. The locations of the approximately 5.6 kb HindIII-SacI fragment subcloned in plasmid LIG 1-3/SAC+MoB2 1SAC, and the SCR coding region are indicated below the restriction map. The location of the translational initiation site of the SCR gene is at the Nco I site at the left end of the indicated coding region. The SCR coding sequence begins at the translation initiation site and extends approximately 1955 nucleotides to its right. E. coli DH5xcex1 containing plasmid pLIG1-3/SAC+MoB2 1SAC, has the ATCC accession number 98031.
FIGS. 15A-S. Comparison of the partial and complete amino acid sequences of several plant members of the SCARECROW family of genes. The amino acid sequences are aligned in a manner that maximizes amino acid sequence similarity and identity among SCR family members. Each sequence shown is continuous except where noted otherwise; the dots are inserted between two sequence segments in order to align homologous segments. xe2x80x9cXxe2x80x9d in the middle of a sequence indicates ambiguity in the corresponding nucleotide sequence and, possible termination of the ORF at the xe2x80x9cXxe2x80x9d residue site. xe2x80x9cXxe2x80x9d at the end of a sequence indicates termination of the ORF at the xe2x80x9cXxe2x80x9d residue site. The numbering of the amino acid residues is shown at the bottom of each figure and is based on the Arabidopsis SCR amino acid sequence. Conserved Motifs I through VI are indicated by the various dashed lines above the figures. The new and old names of the family members are shown in FIG. 15A. The sequences of SCR, Tf1 and Tf4 are of the complete SCR protein. See Table 1 for the identity and the sequence identifier number of each sequence shown in these figures.
FIGS. 16A-M. The partial nucleotide sequences of several plant members of the SCARECROW family of genes. xe2x80x9cNxe2x80x9d indicates an unknown base. See Table 1 for the identity and the sequence identifier number of each sequence shown in these figures.
FIG. 17A. The partial nucleotide sequence (SEQ ID NO:66) of the maize ZCR gene.
FIG. 17B. The partial amino acid sequence (SEQ ID NO:67) of the maize ZCR gene. The underlined sequence shares approximately 80% sequence identity with a corresponding sequence of Arabidopsis SCR protein.
FIG. 18. Comparison of the partial amino acid sequences of several SCR ortholog sequences amplified from the genomes of carrot, soybean and spruce. The SRPd1 and SRPp1 sequences each were obtained by PCR amplification using a combination of 1F and 1R primers. The SRPg1 sequence was obtained by PCR amplification using a combination of 1F and WP primers. The amino acid sequences are aligned in a manner that maximizes amino acid sequence identity and similarity amongst these sequences. Each sequence shown is continuous except where noted otherwise; the dashes are inserted between two sequence segments in order to allow alignment of homologous segments. xe2x80x9cxxe2x80x9d in the middle of a sequence indicates ambiguity in the corresponding nucleotide sequence and, possible termination of the ORF or existence of an intron at the xe2x80x9cxxe2x80x9d residue site. See Table 1 for the identity and the sequence identifier number of each sequence shown in this figure.
FIGS. 19A-G. Comparison of promoter activities in transgenic lines and roots. Panel a. A stably transformed line containing four copies of the B2 subdomain of the 35S promoter of CaMV upstream of GUS (Benfey et al., 1990). GUS is expressed in the root tip. Panel b. Roots emerging from callus transformed with four copies of the B2 subdomain of the 35S promoter fused to GUS. GUS expression can be seen in the emerging root tips (arrows). Panel c. Higher magnification of a root emerging from the callus in panel b. GUS is clearly restricted to the root tip. The morphology of roots regenerated from calli often appears abnormal. Panel d. A transgenic plant regenerated from the calli and roots shown in panel b. GUS expression in this plants appears to be similar to that of the original line shown in panel a. Panel e. ET199, a stably transformed line that contains an enhancer trapping construct with a minimal promoter fused to the GUS coding region inserted 1 kb upstream from the SCR coding region. GUS expression is primarily in the endodermal layer of the root. Panel f. Roots emerging from calli transformed with the SCR promoter::GUS construct. Expression of the GUS gene appears to be limited to an internal layer (arrows). Panel g. SCR promoter::GUS transformed root in liquid culture. Roots shown in panel f were excised and transferred to liquid cultures. GUS expression is primarily found in the endodermal layer as in ET199. The expression of GUS in the quiescent center, as seen here, is also sometimes observed in ET199. Bar, 50 xcexcm.
FIGS. 20A-B. Analysis of SCR promoter activity in the scr mutant background. Panel a. Roots emerging from scr calli transformed with the SCR promoter::GUS construct. Roots regenerated from scr calli are very short. GUS expression appears to be limited to an internal layer of the root (arrows). Panel b. Root regenerated from transformed scr calli and transferred to liquid culture. The scr phenotype, a single layer between the epidermis and pericycle, is easily seen. GUS expression is limited to this mutant layer. E, Epidermis. M, Mutant Layer. P, Pericycle. Bar, 50 xcexcm.
FIGS. 21A-F. Molecular Complementation of the scr mutant. Panels a, c and e. scr transformed with the SCR promoter::GUS construct. Panels b, d and f. scr transformed with the SCR promoter::SCR coding region construct. Panels a and b. Roots emerging from scr calli. Arrows point to several very short roots among many fine root hairs in the scr calli transformed with the SCR promoter::GUS construct. In contrast, roots from scr calli transformed with the SCR promoter::SCR coding region construct appeared to be wild-type in length, suggesting molecular complementation by the transgene. Panels c and d. Transgenic roots in liquid culture. The scr roots transformed with the SCR promoter::GUS construct appeared short, while those transformed with the SCR promoter::SCR coding region construct appeared of wild-type length. Panels e and f. Transverse sections through roots emerging from calli. Whereas there is only a single cell layer between the epidermis and stele in the SCR promoter::GUS transformed root, the radial organization of the root transformed with the SCR promoter::SCR coding region appeared identical to wild-type, with both cortex and endodermal layers. E, epidermis. M, mutant layer. C, cortex. En, Endodermis. P, Pericycle. Bar, 50 xcexcm.
FIG. 22. Expression of ZCR in maize root tips. Left Panel. Expression of ZCR is in the endodermal layer and extends down through the region of the quiescent center. Right Panel. Higher magnification showing expression in a single cell layer through the quiescent center.